PROJECT SUMMARY Cryptosporidium parasites, most commonly Cryptosporidium hominis and Cryptosporidium parvum, cause chronic diarrhea in people with AIDS, and are the second most common cause of life threatening diarrhea in children under one year of age. The currently available drug treatments for immunocompromised people and young children with cryptosporidiosis are poor, and drug development is complicated by a very rudimentary experimental system in which Cryptosporidium cannot even be continually cultivated in vitro. This project addresses both the need for new drug leads and the need for a continuous culture system. The hypothesis is that Cryptosporidium asexual-to-sexual stage differentiation can be targeted to develop both new treatments and tool compounds that enable continuous in vitro cultivation of the asexual forms of the parasite. This hypothesis is based on two facts: 1) that both the asexual and sexual replication cycles of Cryptosporidium occur in a single host with oocyst sporulation occurring in situ, which yields immediately infectious oocysts leading to chronic auto-infection in compromised patients; and 2) that, at least in vitro, the asexual type I meront forms of the parasite all develop into late-stage type II meronts that are committed to undergoing sexual differentiation. Interference with sexual differentiation at late-stages is predicted to block auto-infection and result in cure, and interference with differentiation at early-stages is predicted to prevent differentiation to the committed, type II meront and enable continuous culture of the asexual forms. Data describing a novel biomarker, a high content imaging assay method for C. parvum sexual differentiation, and identification of one differentiation inhibitor are presented. Additional inhibitors will be identified in aim1 by screening a library of biologically active, drug-like compounds. In aim 2, compounds will be classified as potentially useful as drug development starting points or as tool compounds to achieve culture synchronization and/or continuous culture. The effects of compounds on Cryptosporidium will then be characterized using transmission electron microscopy, a C. parvum tissue culture development assay, an in vitro pharmacodynamics assay, and a NOD SCID gamma mouse model of chronic cryptosporidiosis. Completion of these studies is expected to determine the utility of targeting Cryptosporidium differentiation, to yield well characterized small molecules drug leads and tool compounds, and, if the hypothesis is correct, to yield a simple method for continual cultivation of Cryptosporidium.